Alkaliphilic Bacillus pseudofirmus OF4 grows at pH e 11.2, but the transmembrane electrochemical gradient of protons, the bulk Protonmotive Force (PMF) that energizes ATP synthesis, is too low to account for the ATP produced. It is too low because, by necessity, the alkaliphile uses a very effective cation/proton antiporter (exchanger) type, a multi-subunit Mrp antiporter, to maintain a cytoplasmic pH that is as much as 2.3 pH units lower than the external pH. Findings of ATP synthesis at low PMF and new probes that can track protons pumped by the respiratory chain, contributed to a recent paradigm shift, a new consensus that protons emerging on the membrane surface are retained there long enough to reach an ATP synthase before fully equilibrating with the bulk medium, i.e. energization is by a surface PMF. Interfacial water barriers and charge distribution on the membrane surface are hypothesized contributors to proton retention. The dwell time on the membrane surface also makes pumped protons accessible to membrane proteins that could enhance ATP synthesis by creating domains in which the pumps and synthase are in closer proximity. Similarly, outer membrane constituents (in mitochondria or Gram-negative bacteria) or cell wall associated polymers, e.g. Secondary Cell Wall Polymers (in Gram-positive bacteria) may contribute to proton retention by delaying proton loss from the periplasm. With the whole pharmacology of ATP synthesis changing in this new paradigm, we will systematically use knock-outs to explore roles of Secondary Cell Wall Polymers and Flotillin- related membrane proteins in ATP synthesis efficacy in the alkaliphile model system. Earlier findings of motifs in the alkaliphile c-rotor of ATP synthase that promote synthesis, will inform new studies of the catalytic properties of Staphylococcus aureus ATP synthase and the structure/function properties of its c-subunit rotor, which are recognized potential antibiotic targets in this major pathogen. The Mrp-type antiporters play crucial roles in S. aureus as well as in a large group of other pathogens that thrive in non-fermentative high energization contexts. Structural studies will be initiated on the alkaliphile Mrp antiporter, which could provide invaluable information for inhibitor design. The ful cohort of S. aureus antiporters, including two Mrp-type antiporters and six others from three different antiporter groups, will also be characterized with respect to their activity profiles, expression levels under diverse conditions and the effect of their deletion on S. aureus growth under a matrix of conditions. This will identify additional antiporters with target potntial and fill a knowledge gap that impedes our ability to model the patho-physiological behavior of S. aureus. RELEVANCE: The importance of proton retention at the membrane surface brings membrane properties such as stiffness vs. leakiness into stronger relationship with obesity vs. leanness for mitochondria or properties of bacterial membranes into closer connection with robust vs. weaker energy generation serving pathogenesis.